Alp conjugate substrate kit

T cell memory responses from immunized splenocytes were quantified using enzyme-linked immunospot (ELISpot) assay. Multiscreen-IP filter plates (MSIPS4510, Merck) were coated with anti-mouse IFNγ antibody (5 μg/mL, 100 μL/well, clone AN18, MABTech, Nacka Strand, Sweden) or anti-mouse IL4 antibody (5 μg/mL, 100 μL/well, BVD4-1D11, BD Biosciences) overnight at 4 °C, washed 5 times with 200 μL/well sterile PBS and blocked with complete RPMI (150 μL/well) for a minimum of 1 h at 37 °C. Then, 15 μg/mL of MSP4/5 was used for recall with 5 × 10⁵ splenocytes. Medium alone was used as the negative control and 1 μg/mL Concavalin A as a positive control. IFNγ was labelled with biotin anti-mouse IFNγ antibody (1 μg/mL, 100 μL/well, R4-6A2-Biotin, MABTech) or biotin anti-mouse IL4 (1 μg/mL, 100 μL/well, BVD6-24G2, BD Biosciences) for 2 h at room temperature. This was then labelled with streptavidin-alkaline phosphatase (ALP) (1:1000, 100 μL/well, 3310-10, MABTech) or ExtrAvidin-ALP (1:3000, 100 μL/well, Sigma-Aldrich) for 1.5 h at room temperature. Spots were developed with an ALP conjugate substrate kit (Bio-Rad, Hercules, CA, USA) and imaged and counted with an ELISpot reader and software (AID, Strasburg, Germany).